The present invention relates in general to an apparatus and a method for use in electron microscopy, and more particularly, to an apparatus and a method including a direct bombardment detector and a secondary detector for use in electron microscopy.
Conventional electron microscopy use either photographic film or electronic image sensor based cameras to detect high-energy charged particles directly or indirectly with an intervening charged-particle-sensitive scintillator screen. The scintillator screen converts the impinging high-energy charged particle image into an image that can be collected on photon sensitive devices. These existing detection techniques have several drawbacks, such as limited sensitivity, limited resolution, poor usability, and time inefficiency.
Electron microscopy (EM) has been applied in various fields with the benefit of providing images at higher sensitivity, improved resolution, and a more timely fashion. One specific type of electron microscopy, that is, transmission electron microscopy (TEM) has been applied for the analysis of protein structure. X-ray protein crystallography is currently the premier method to determine the three-dimensional structures of large proteins such as DNA, RNA/protein complexes, and viruses. Cryo-electronic microscopy (cryo-EM) is emerging as a technique complementary to X-ray protein crystallography. An advantage of cryo-EM over X-ray protein crystallography is that it does not depend on the time-consuming, challenging, and in some case impossible process for growing crystals of the material to be studied. Despite the resolution limitations, cryo-EM has been used successfully in time-resolved experiments to reveal the dynamic aspects of protein interaction, something not possible in a crystalline material.
Three principal factors limit the resolution of the structures characterized by the cryo-EM. The first is the difficulties with specimen stabilization and radiation damage. The second is the difficulties of collection and processing of very large data sets required for statistical analysis. The third is the aberrations in the electron image (electron microscopy signal) formed in the microscope. The third factor has been essentially overcome recently. Instrumentation features have been developed to essentially remove chromatic aberration. The problem with the specimen stabilization and radiation damage has been partially resolved with electron microscopes that feature exceptional coherence and stability and the cryogenic techniques. The radiation damage, though mitigated, remains as a serious problem in application of electron microscope. Many materials of interest are extremely dose-sensitive. The high microscope magnification and sample dose required to acquire images of sufficient resolution and signal-to-noise ratio (SNR) often disrupt the sample. Data sets acquired with less than optimal quality are inevitably used.
The use of film to record images in electron microscopy, particularly in cryo-EM, is problematic. The film provides excellent modulation transfer and a large field of view, but requires several cumbersome post-acquisition steps. For example, the film must be processed to develop the latent image and scanned for digitization. These steps are time-consuming and require additional facilities and equipment, which results in a lack of immediacy for which digital cameras are known. The use of film can be difficult even in the pre-acquisition stage. For example, it is time-consuming to load or unload the film to and from a typical transmission microscope because it requires breaking and re-establishing microscope vacuum. Both the pre- and post-acquisition steps have the risk of fogging the film and misleading that leads to jams and further delays.
Using film for cryo-EM is particularly daunting. As estimated, one may need to collect up to 100,000 images for getting a 10 Å resolution structure of a large protein complex like the ribosome. Similarly, a 3 Å resolution structure of a structure would require up to 1 million images with the application of film, which renders the detection technique highly impractical.
Charged coupled device detectors (CCDs) have been increasingly used for detection in electron microscopy. These detectors overcome the time-consuming steps of loading, unloading, processing, and digitizing by providing a digital output directly. Commonly available CCD detectors have formats up to 4096 by 4096 pixels (4K×4K), although they fall short of delivering the full resolution anticipated by the pixel count alone. The CCD detectors and other silicon image sensors such as CMOS image sensors require the use of a fluorescent scintillation screen to convert the charged particle image to a photon image within the range where the detector efficiency is the highest. Unfortunately, with each charged-particle event, the size of a fluorescent spot produced within the scintillation screen is larger than the detector pixel size. Although scintillator material layer thickness can be reduced to reduce spot size, sensitivity is sacrificed as the number of photons produced per incident primary electron is also reduced. At 300 KeV, the full width at half maximum of the spot from a typical scintillator material is about 30 μm. However, the full width at 1% is 200 μm. The large spread of light reduces the effective resolution of typical commercial CCD cameras by at least a factor of two in each dimension rendering the resolution of a 4K CCD camera reduced to that of a 2K×2K camera only.
Fiber optic coupling between the CCD and the scintillator is the most common current design because it maximizes light collection efficiency. However, it introduces additional light scattering which further degrades the transfer of spatial information. Any material placed directly behind the scintillator layer can also produce scattering of the primary electrons. Some of the scattered electrons can re-enter the scintillator layer to produce a secondary spot of light displaced from the first that also degrades resolution. Additionally, the high-energy electrons induce damage in the scintillator and fiber optic degrading performance and ultimately requiring their replacement. All of these effects become more severe at the higher accelerating voltages desirable for cryo-EM work. In spite of these limitations, straight fiber optic bundle coupled cameras are now the most widely used detectors in electron microscopy.
To mitigate the loss in resolution resulting from the sever mismatch between the detector pixel size and the scintillator spot size, tapered fiber optics and demagnification lens systems have been developed. Tapered fiber optic coupling introduces additional large spatial distortions and non-uniformities over those present in straight fiber optic couplings. The additional distortions are difficult to correct mathematically. As a result, the application of this type of fiber optic is less widely used.
Lens systems that provide image demagnification sacrifice light gathering efficiency to preserve spatial information transfer. For sensitive samples where low electron doses are required, light gathering efficiency must be maximized by using very high numerical aperture lenses. Image de-magnifying lens transfer systems that maximize light collection efficiency are large and expensive.
A new type of detector for electron microscopy is disclosed in U.S. Pat. No. 7,262,411 entitled “Direct Collection Transmission Electron Microscopy” the complete contents of which are incorporated herein by reference. Active sensors are used in direct bombardment mode to achieve direct detection without use of a film or a scintillator screen. The new detector comprises a plurality of active pixel sensors each containing a photodiode that collects secondary electrons generated when a primary electron passes through the epitaxial silicon layer in which the p-n junction of the photodiode is formed. These detectors achieve high-speed readout, high resolution, and very high sensitivity to single primary electrons. While these devices are specifically designed to withstand the rigors of direct bombardment, like fiber optic bundles, they eventually suffer damages and/or degradation.
Accordingly, there exists a need in the art for an improved apparatus and/or method for imaging a sample in electron microscopy.